In the US, herpes simplex virus type 1 (HSV-1) is the most common cause of corneal blindness due to an infectious agent and the most common cause of sporadic lethal encephalitis in immune competent individuals. Most HSV-1 disease is due to viral reactivations rather than the primary acute infection. Thus understanding HSV-1 latency-reactivation is important for developing methods to combat HSV-1 diseases. HSV-1 uses herpes virus entry mediator, or HVEM, as one route of infecting cells. HVEM is a member of the TNF receptor superfamily and can regulate immune responses by acting as a molecular switch between proinflammatory and inhibitory signaling. When HVEM on a neuron binds to BTLA on a T cell, a bidirectional pathway is activated. Activated HVEM activates NF-kB which promotes neuronal cell survival by decreasing apoptosis. Activated BTLA limits T cell activation. Thus, increasing HVEM should block apoptosis and decrease T cell function - the same activities attributed to the HSV-1 LAT gene and that are thought to be involved in how LAT enhances latency/reactivation (although the mechanism(s) by which LAT blocks apoptosis and decreases T cell response are not known). Our preliminary and recently published results indicate that: 1)HSV-1 latency and reactivation are both significantly reduced in HVEM-/- mice and 2)Two small non-coding LAT RNAs (sncRNAs) can upregulate HVEM on neurons. Based on these findings we hypothesize that one mechanism by which LAT enhances latency and reactivation is by upregulating HVEM which in turn promotes HSV-1 latency and reactivation by decreasing both apoptosis and the local T cell response. Our Specific Aims are: 1. Construct an HSV-1 double knockout (KO) mutant (DsncRNA1&2) that is KO'd for both LAT sncRNAs (sncRNA1 & sncRNA2). We expect that this mutant will no longer increase HVEM expression and will have reduced latency/reactivation similar to LAT(-) mutants. This would strongly support the hypothesis that the two LAT sncRNAs normally upregulate HVEM which in turn decreases apoptosis and T cell responses resulting in increased latency/reactivation. 2. Construct and analyze an HSV-1 mutant (DLAT-HVEM) that expresses HVEM driven by the LAT promoter on a LAT(-) genomic background. We expect the HVEM expressed by this mutant to restore wt LAT(+)- like latency/reactivation to the LAT(-) virus. This would strongly support the hypothesis that increasing HVEM expression is an important mechanism by which LAT enhances latency/reactivation. We expect successful completion of this exploratory R21 application to lead to an RO1 grant involving the mechanism of LAT upregulation of HVEM and the development of therapeutic interventions to block this upregulation or block components of the bidirectional HVEM pathway.